Stilbenoids are biologically active phenolic compounds that exhibit a wide spectrum antibiotic and pharmacological activity.
It is known that certain plants, such as grapevine, can synthesize stilbenoids as an adaptative mechanism in response to a stress, such as ultraviolet irradiation or a microbial infection (Derks et al. 1995. “Stilbene phytoalexins and disease resistance in Vitis”. In Handbook of Phytoalexin Metabolism and Action, M. Daniel and R. P. Purkayastha, eds., Marcel Dekker, Inc. USA, pp. 287-315). One of the main constituents of these compounds is trans-resveratrol or t-resveratrol (trans-3,5,4′-trihydroxystilbene) (Langcake, P. and Pryce, R. J. 1976. “The production of resveratrol by Vitis vinifera and other members of the Vitaceae as a response to infection or injury”, Physiol. Plant. Pathol. 9:77-86).
The cis-resveratrol isomer is usually present in extracts from plants producing t-resveratrol and derivative products. Its presence is due to the slow photoisomerization of the trans isomer by irradiation with ultraviolet light (Mattivi, F. et al. 1995. “Isolation, characterization and evolution in red wine vinification of resveratrol monomers” J. Agric. Food Chem. 43:1820-1823).
Based on epidemiological and laboratory studies involving humans, animals, animal cells in culture, and enzyme assays, it has been demonstrated that stilbenoids, and particularly resveratrol, have favourable effects on health (Jang, et al. 1997. “Cancer chemopreventive activity of resveratrol, a natural product derived from grapes”, Science 275:218-220). Therefore, inclusion in human and animal diet of ingestible products containing resveratrol is desirable.
Resveratrol is present in wine and may be implicated in the salutary effects of moderate wine consumption. An increased resveratrol consumption has been proposed as a way for reducing the incidence of cancer and cardiovascular diseases in humans (Soleas, et al. 1997. “Wine as a biological fluid: history, production, and role in disease prevention”, J. Clin. Lab. Anal. 11:287-313). Resveratrol and plant extracts containing resveratrol have been shown to be effective for the prevention and treatment of arteriosclerosis (Arichi, et al. 1982. “Effect of stilbene components of the roots of Polygonum cuspidatum Sieb. et Zucc. on lipid metabolism” Chem. Pharm. Bull. 30:1766-1770), as an anti-inflammatory agent (Kimura, et al. 1985. “Effects of stilbenes on arachidonate metabolism in leukocytes” Biochem. Biophys Acta 834:275-278), and as an anti-hyperoxidative agent (Kimura et al. 1983. “Effects of stilbene components of roots of Polygonum ssp. on liver injury in perodized oil-fed rats” Planta Medica. 49: 51-54). Resveratrol has shown a significant inhibition of crypt formation in aberrant colon in a rat model treated with a carcinogenic agent (azoxymethane), thus suggesting its value as a tumour generation inhibitor in humans (Steele et al. 1998. “Cancer chemoprevention drug development strategies for resveratrol”, Pharm. Bio. 36:62-68 suppl.). Resveratrol has also been reported to promote the formation of nitroxides, which are vasodilating agents and anti-platelet aggregants (Fitzpatrick et al. 1993. “Endothelium-dependent vasorelaxing activity of wine and other grape products” Am. J. Physiol. 265: H774-H778).
Considering the beneficial role of resveratrol on human and animal health, it is important to have available an adequate biological source that allows for obtaining resveratrol in adequate amounts to meet the demand. Various studies have been conducted for this purpose.
In a study, whole grapevine plants were treated with aluminium chloride, which acts as an agent eliciting resveratrol synthesis, to increase resveratrol content in the plant and its derivative products, such as grapes, must, and wine (Jeandet et al. “Utilisation du chlorure d'aluminium comme agent éliciteur de la synthése du resvératrol”, patent application WO97/18715).
In another study, the resveratrol synthase gene, or a portion thereof, was transferred to a plant not naturally producing resveratrol so that the plant constitutively expresses the gene and accumulates the derivative resveratrol glucoside in its tissues (Hipskind, J. D. “Transgenic plants modified to contain resveratrol glucoside and uses thereof”, patent application WO00/44921).
In another study, suspensions of cells from plants producing resveratrol were elicited with portions of fungal cell walls to induce resveratrol synthesis and its accumulation in the culture medium and cells (Liswidowati, et al. 1991. “Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells” Planta 183:307-314).
A further study reported the ability of a cyclodextrin, specifically heptakis-(2,6-di-O-methyl-β-cyclodextrin) (DIMEB), to induce in grapevine cell suspensions (Vitis vinifera var. Gamay) the synthesis of t-resveratrol, which was excreted into the culture medium (Morales et al. 1998. “Effect of dimethyl-β-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before and after inoculation with Xylophilus ampelinus”, Plant Cell Tiss. Org. Cult. 53:179-187). Cyclodextrins are already known to share the property of increasing the aqueous solubility of poorly water soluble compounds by forming inclusion complexes. Inclusion complexes are formed when a host molecule is housed in the central cavity of the cyclodextrin molecule, the assembly having a similar solubility to free cyclodextrin (Saenger, W. 1980. “Cyclodextrin inclusion compounds in research and industry”, Angew. Chem. Int. Ed. Engl. 19:344-362). Based on these properties, t-resveratrol excreted by cells can form inclusion complexes with cyclodextrins, and may accumulate in the culture medium at concentrations higher than its aqueous solubility limit without precipitating.
However, various tests conducted by the inventors have demonstrated that, contrary to what could be expected, not all cyclodextrins have the ability to act as elicitors of resveratrol synthesis in cell cultures, and further research is still required in this field.